We previously determined that a region between positions -228 and -189 upstream from the human gamma-globin genes can form an intramolecular triplex (H-DNA) in supercoiled plasmids. To identify proteins that might interact with this DNA structure, we performed expression cloning using an adult bone marrow cDNA library and the single-stranded region of the H-DNA structure as a probe. We cloned molecules very similar to two previously identified cDNAs, dbpA and dbpB. The dbpB-like protein (called BP-8 in this study) interacts specifically (KD approximately 4 nM) with two homopyrimidine "half-sites" in the single-stranded gamma-228 to -189 probe, but binds to double-stranded DNA containing the same sequence with 100-fold less affinity. We have also shown that supercoiled plasmids containing the gamma-228 to -189 region contain a high affinity binding site for BP-8 that is stabilized by factors that stabilize H-DNA; two HPFH point mutations (-202 C-->G or C-->T) that destabilize the secondary DNA structure abolish the high affinity binding site. Collectively, these data show that dbpB/BP-8 binds specifically to homopyrimidine half-sites in single-stranded DNA, and that it also binds to H-DNA structures that contain homopyrimidine tracts in the single-stranded and triple-stranded regions.